Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.

The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.

Prion Protein-Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering.

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP(C)) into a ß-sheet-rich disease-causing isoform (PrP(Sc)) is the key molecular event in the formation of PrP(Sc) aggregates. The molecular mechanisms underlying the PrP(C)-to-PrP(Sc) conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP(Sc) in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23-230)] and N-terminally truncated recPrP(89-230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP(C) into PrP(Sc).

The origin of the electrostatic perturbation in acetoacetate decarboxylase.

Acetoacetate decarboxylase (AADase) has long been cited as the prototypical example of the marked shifts in the pK(a) values of ionizable groups that can occur in an enzyme active site. In 1966, it was hypothesized that in AADase the origin of the large pK(a) perturbation (-4.5 log units) observed in the nucleophilic Lys 115 results from the proximity of Lys 116, marking the first proposal of microenvironment effects in enzymology. The electrostatic perturbation hypothesis has been demonstrated in a number of enzymes, but never for the enzyme that inspired its conception, owing to the lack of a three-dimensional structure. Here we present the X-ray crystal structures of AADase and of the enamine adduct with the substrate analogue 2,4-pentanedione. Surprisingly, the shift of the pK(a) of Lys 115 is not due to the proximity of Lys 116, the side chain of which is oriented away from the active site. Instead, Lys 116 participates in the structural anchoring of Lys 115 in a long, hydrophobic funnel provided by the novel fold of the enzyme. Thus, AADase perturbs the pK(a) of the nucleophile by means of a desolvation effect by placement of the side chain into the protein core while enforcing the proximity of polar residues, which facilitate decarboxylation through electrostatic and steric effects.

Trapping and structure determination of an intermediate in the allosteric transition of aspartate transcarbamoylase.

X-ray crystallography and small-angle X-ray scattering (SAXS) in solution have been used to show that a mutant aspartate transcarbamoylase exists in an intermediate quaternary structure between the canonical T and R structures. Additionally, the SAXS data indicate a pH-dependent structural alteration consistent with either a pH-induced conformational change or a pH-induced alteration in the T to R equilibrium. These data indicate that this mutant is not a model for the R state, as has been proposed, but rather represents the enzyme trapped along the path of the allosteric transition between the T and R states.

Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect.

Helicobacter pylori NikR (HpNikR) is a nickel-dependent transcription factor that regulates multiple genes in the H. pylori pathogen. There are conflicting data regarding the locations of the Ni(II) sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (i) the metal-binding domain (MBD) of HpNikR (3.08 Å) and (ii) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-HpNikR compared with the affinities of Ni(II) reconstituted apo-HpNikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-HpNikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by HpNikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata.

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.

An integrated high-throughput data acquisition system for biological solution X-ray scattering studies.

A fully automated high-throughput solution X-ray scattering data collection system has been developed for protein structure studies at beamline 4-2 of the Stanford Synchrotron Radiation Lightsource. It is composed of a thin-wall quartz capillary cell, a syringe needle assembly on an XYZ positioning arm for sample delivery, a water-cooled sample rack and a computer-controlled fluid dispenser. It is controlled by a specifically developed software component built into the standard beamline control program Blu-Ice/DCS. The integrated system is intuitive and very simple to use, and enables experimenters to customize data collection strategy in a timely fashion in concert with an automated data processing program. The system also allows spectrophotometric determination of protein concentration for each sample aliquot in the beam via an in situ UV absorption spectrometer. A single set of solution scattering measurements requires a 20-30 µl sample aliquot and takes typically 3.5 min, including an extensive capillary cleaning cycle. Over 98.5% of measurements are valid and free from artefacts commonly caused by air-bubble contamination. The sample changer, which is compact and light, facilitates effortless switching with other sample-handling devices required for other types of non-crystalline X-ray scattering experiments.

Characterization of lipid matrices for membrane protein crystallization by high-throughput small angle X-ray scattering.

The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) - the most direct technique to identify lipid mesophases and measure their structural parameters - to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments.

Balanced electrostatic and structural forces guide the large conformational change associated with maturation of T = 4 virus.

Nudaurelia capensis omega virus has a well-characterized T = 4 capsid that undergoes a pH-dependent large conformational changes (LCC) and associated auto-catalytic cleavage of the subunit. We examined previously the particle size at different pH values and showed that maturation occurred at pH 5.5. We now characterized the LCC with time-resolved small-angle x-ray scattering and showed that there were three kinetic stages initiated with an incremental drop in pH: 1), a rapid (<10 ms) collapse to an incrementally smaller particle; 2), a continuous size reduction over the next 5 s; and 3), a smaller final transition occurring in 2-3 min. Equilibrium measurements similar to those reported previously, but now more precise, showed that the particle dimension between pH 5.5 and 5 requires the autocatalytic cleavage to achieve its final compact size. A balance of electrostatic and structural forces shapes the energy landscape of the LCC with the latter requiring annealing of portions of the subunit. Equilibrium experiments showed that many intermediate states could be populated with a homogeneous ensemble of particles by carefully controlling the pH. A titration curve for the LCC was generated that showed that the virtual pK(a) (i.e., the composite of all titratable residues that contribute to the LCC) is 5.8.

Virus capsid expansion driven by the capture of mobile surface loops.

The capsids of tailed-DNA bacteriophages first assemble as procapsids, which mature by converting into a new form that is strong enough to contain a densely packed viral chromosome. We demonstrate that the intersubunit crosslinking that occurs during maturation of HK97 capsids actually promotes the structural transformation. Small-angle X-ray scattering and crosslinking assays reveal that a shift in the crosslink pattern accompanies conversion of a semimature particle, Expansion Intermediate-I/II, to a more mature state, Balloon. This transition occurs in a switch-like fashion. We find that crosslink formation shifts the global conformational balance to favor the balloon state. A pseudoatomic model of EI-I/II derived from cryo-EM provides insight into the relationship between crosslink formation and conformational switching.

A covalent linker allows for membrane targeting of an oxylipin biosynthetic complex.

A naturally occurring bifunctional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C-terminal lipoxygenase (LOX) portion of the molecule catalyzes the transformation of arachidonic acid (AA) to the corresponding 8 R-hydroperoxide, and the N-terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). Small-angle X-ray scattering data indicate that in the absence of a covalent linkage the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bifunctional protein. The SAXS data also support a model for LOX and AOS domain orientation in the fusion protein inferred from a low-resolution crystal structure. However, results of membrane binding experiments indicate that covalent linkage of the domains is required for Ca (2+)-dependent membrane targeting of the sequential activities, despite the noncovalent domain association. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and LOX domains. Our data are consistent with a model in which Ca (2+)-dependent membrane binding relieves the noncovalent interactions between the AOS and LOX domains and suggests that the C2-like domain of LOX mediates both protein-protein and protein-membrane interactions.

Use of L-asparagine and N-phosphonacetyl-L-asparagine to investigate the linkage of catalysis and homotropic cooperativity in E. coli aspartate transcarbomoylase.

The mechanism of domain closure and the allosteric transition of Escherichia coli aspartate transcarbamoylase (ATCase) are investigated using L-Asn, in the presence of carbamoyl phosphate (CP), and N-phosphonacetyl-L-asparagine (PASN). ATCase was found to catalyze the carbamoylation of L-Asn with a K(m) of 122 mM and a maximal velocity 10-fold lower than observed with the natural substrate, L-Asp. As opposed to L-Asp, no cooperativity was observed with respect to L-Asn. Time-resolved small-angle X-ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and L-Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K(D) of 8.8 microM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme. The linking of CP and L-Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of L-Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high-activity high- affinity R structure. These results imply that Arg229, which interacts with the beta-carboxylate of L-Asp, plays a critical role in the orientation of L-Asp in the active site and demonstrates the requirement of the beta-carboxylate of L-Asp in the mechanism of domain closure and the allosteric transition in E. coli ATCase.

Gain-of-function mutational activation of human tRNA synthetase procytokine.

Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase.

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.

Structure of the E.coli aspartate transcarbamoylase trapped in the middle of the catalytic cycle.

Snapshots of the catalytic cycle of the allosteric enzyme aspartate transcarbamoylase have been obtained via X-ray crystallography. The enzyme in the high-activity high-affinity R state contains two catalytic chains in the asymmetric unit that are different. The active site in one chain is empty, while the active site in the other chain contains an analog of the first substrate to bind in the ordered mechanism of the reaction. Small angle X-ray scattering shows that once the enzyme is converted to the R state, by substrate binding, the enzyme remains in the R state until substrates are exhausted. Thus, this structure represents the active form of the enzyme trapped at two different stages in the catalytic cycle, before the substrates bind (or after the products are released), and after the first substrate binds. Opening and closing of the catalytic chain domains explains how the catalytic cycle occurs while the enzyme remains globally in the R-quaternary structure.

Cooperative reorganization of a 420 subunit virus capsid.

The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, large-scale conformational maturation of the 420 subunit, 13 MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60 A larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles "hop" over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.

A single amino acid substitution in the active site of Escherichia coli aspartate transcarbamoylase prevents the allosteric transition.

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side-chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved, small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7 angstroms resolution. Time-resolved, small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side-chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side-chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate-binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low-activity low-affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate-binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase.

N-phosphonacetyl-L-isoasparagine a potent and specific inhibitor of Escherichia coli aspartate transcarbamoylase.

The synthesis of a new inhibitor, N-phosphonacetyl-L-isoasparagine (PALI), of Escherichia coli aspartate transcarbamoylase (ATCase) is reported, as well as structural studies of the enzyme.PALI complex. PALI was synthesized in 7 steps from beta-benzyl L-aspartate. The KD of PALI was 2 microM. Kinetics and small-angle X-ray scattering experiments showed that PALI can induce the cooperative transition of ATCase from the T to the R state. The X-ray structure of the enzyme.PALI complex showed 22 hydrogen-bonding interactions between the enzyme and PALI. The kinetic characterization and crystal structure of the ATCase.PALI complex also provides detailed information regarding the importance of the alpha-carboxylate for the binding of the substrate aspartate.